Antisense polynucleotide

ABSTRACT

The present invention discloses methods for the treatment of colon cancer. The expression of gastrin by colon cancers is inhibited by the use of antisense gastrin expression. Methods are disclosed for the preparation of expression constructs and the use of such constructs to inhibit colon cancer growth.

The U.S. government owns rights in the present invention pursuant togrant numbers CA60087 from the National Institutes of Health.

This application is a continuation-in-part of U.S. Ser. No. 08/634,546filed Apr. 18, 1996, now U.S. Pat. No. 5,786,213.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to treatment of colon cancers.More particularly, it concerns the use of antisense gastrin expressionto reduce colon cancer growth.

2. Description of Related Art

Colorectal cancers are among the most common cancers in men and women inthe U.S. and are one of the leading causes of death (Steel, 1994). Otherthan surgical resection no other systemic or adjuvant therapy isavailable. Vogelstein and colleagues have described the sequence ofgenetic events that appear to be associated with the multistep processof colon cancer development in humans (Fearon and Vogelstein, 1990). Anunderstanding of the molecular genetics of carcinogenesis, however, hasnot led to preventative or therapeutic measures. It can be expected thatadvances in molecular genetics will lead to better risk assessment andearly diagnosis but colorectal cancers will remain a deadly disease fora majority of patients due to the lack of an adjuvant therapy. Adjuvantor systemic treatments are likely to arise from a better understandingof the autocrine factors responsible for the continued proliferation ofcancer cells.

Endogenous gastrins and exogenous gastrins (other than tetragastrin)seem to promote the growth of established colon cancers in mice (Singh,et al., 1986; Singh, et al., 1987; et al., 1984; Smith and Solomon,1988; Singh, et al., 1990; Rehfeld and van Solinge, 1994) and promotecarcinogen induced colon cancers in rats (Williamson et al., 1978;Karlin et al., 1985; Lamoste and Willems; 1988). Recent studies ofMontag et al. (1993) further support a possible co-carcinogenic role ofgastrin in the initiation of tumors.

Many colon cancer cells express and secrete gastrin gene products (Daiet al., 1992; Kochman et al., 1992; Finley et al., 1993; Van Solinge etal., 1993; Xu et al., 1994; Singh et al., 1994a; Hoosein et al., 1988;Hoosein et al., 1990) and bind gastrin-like peptides (Singh et al.,1986; Singh et al., 1987; Weinstock and Baldwin, 1988; Watson andSteele, 1994; Upp et al., 1989; Singh et al., 1985). In previous reportsgastrin antibodies were either reported to inhibit (Hoosein et al.,1988; Hoosein et al., 1990) or have no effect on the growth of coloncancer cell lines in vitro.

However other investigators have had inconclusive results with coloncancer cell lines. A number of studies testing the effects of gastrin oncell proliferation of cancer cells have been performed (Sirinek et al.,1985; Kusyk et al., 1986; Watson et al., 1989). The results have variedwidely. In one study, four different human cancer cell lines were testedfor growth stimulation by pentagastrin and only one showed growthstimulation (Eggstein et al., 1991). Similarly, in a majority of thestudies conducted to-date, mitogenic effects of gastrin have beendemonstrated only on a very small percentage of colon cancer cell linesin vitro (Hoosein et al., 1988; Hoosein et al., 1990; Shrink et al.,1985; Kusyk et al., 1986; Guo et al., 1990; Ishizuka et al., 1994).

Since only a small percentage of established human colon cancer celllines demonstrated a growth response to exogenous gastrins,investigators in this field came to believe that gastrin probably didnot play a significant role in the growth of colon cancers. The recentdiscovery that human colon cancer cell lines and primary human coloncancers express the gastrin gene has sparked a renewed interest in apossible autocrine role of gastrin-like peptides in colon cancers.However, significant skepticism remains in the field, to date, regardingthe importance of gastrin gene expression in the continued growth andtumorogenicity of colon cancers.

Thus, to-date, no systemic or adjuvant therapies have been developed forcolon cancers, based on the knowledge that a significant percentage ofhuman colon cancers express the gastrin gene. In fact, no adjuvant orsystemic therapy has been developed for colon cancers that is based onthe knowledge of the expression of other growth factors such as TGFα orIGF-11, since none of the growth factors demonstrate a significantgrowth effect on a majority of the colon cancer cell lines in culture.

At the present time the only systemic treatment available for coloncancer is chemotherapy. However, chemotherapy has not proven to be veryeffective for the treatment of colon cancers for several reasons, themost important of which is the fact that colon cancers express highlevels of the MDR gene (that codes for multi-drug resistance geneproducts). The MDR gene products actively transport the toxic substancesout of the cell before the chemotherapeutic agents can damage the DNAmachinery of the cell. These toxic substances harm the normal cellpopulations more than they harm the colon cancer cells for the abovereasons.

There is no effective systemic treatment for treating colon cancersother than surgically removing the cancers. In the case of several othercancers, including breast cancers, the knowledge of growth promotingfactors (such as EGF, estradiol, IGF-11) that appear to be expressed oreffect the growth of the cancer cells, has been translated for treatmentpurposes. But in the case of colon cancers this knowledge has not beenapplied and therefore the treatment outcome for colon cancers remainsbleak.

Antisense RNA technology has been developed as an approach to inhibitinggene expression, particularly oncogene expression. An "antisense" RNAmolecule is one which contains the complement of, and can thereforehybridize with, protein-encoding RNAs of the cell. It is believed thatthe hybridization of antisense RNA to its cellular RNA complement canprevent expression of the cellular RNA, perhaps by limiting itstranslatability. While various studies have involved the processing ofRNA or direct introduction of antisense RNA oligonucleotides to cellsfor the inhibition of gene expression (Brown, et al., 1989; Wickstrom,et al., 1988; Smith, et al., 1986; Buvoli, et al., 1987), the morecommon means of cellular introduction of antisense RNAs has been throughthe construction of recombinant vectors which will express antisense RNAonce the vector is introduced into the cell.

A principle application of antisense RNA technology has been inconnection with attempts to affect the expression of specific genes. Forexample, Delauney, et al. have reported the use antisense transcripts toinhibit gene expression in transgenic plants (Delauney, et al., 1988).These authors report the down-regulation of chloramphenicol acetyltransferase activity in tobacco plants transformed with CAT sequencesthrough the application of antisense technology.

Antisense technology has also been applied in attempts to inhibit theexpression of various oncogenes. For example, Kasid, et al., 1989,report the preparation of recombinant vector construct employing Craf-1CDNA fragments in an antisense orientation, brought under the control ofan adenovirus 2 late promoter. These authors report that theintroduction of this recombinant construct into a human squamouscarcinoma resulted in a greatly reduced tumorigenic potential relativeto cells transfected with control sense transfectants. Similarly,Prochownik, et al., 1988, have reported the use of Cmyc antisenseconstructs to accelerate differentiation and inhibit G₁ progression inFriend Murine Erythroleukemia cells. In contrast, Khokha, et al., 1989,discloses the use of antisense RNAs to confer oncogenicity on 3T3 cells,through the use of antisense RNA to reduce murine tissue inhibitor ormetalloproteinases levels.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with "complementary" sequences. Complementary sequences arethose polynucleotides which are capable of base-pairing according to thestandard Watson-Crick complementarity rules. That is, the larger purineswill base pair with the smaller pyrimidines to form combinations ofguanine paired with cytosine (G:C) and adenine paired with eitherthymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) inthe case of RNA. Inclusion of less common bases such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal., including a human subject.

Throughout this application, the term "expression vector or construct"is meant to include any type of genetic construct containing a nucleicacid coding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a gene andtranslation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encoding agene of interest.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A "promoter" refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase "under transcriptional control" means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter is used to refer to a group of transcriptional controlmodules that are clustered around the initiation site for RNA polymeraseII. Much of the thinking about how promoters are organized derives fromanalyses of several viral promoters, including those for the HSVthymidine kinase (tk) and SV40 early transcription units. These studies,augmented by more recent work, have shown that promoters are composed ofdiscrete functional modules, each consisting of approximately 7-20 bp ofDNA, and containing one or more recognition sites for transcriptionalactivator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid encoding a particular gene is not believed to be important,so long as it is capable of expressing the nucleic acid in the targetedcell. Thus, where a human cell is targeted, it is preferable to positionthe nucleic acid coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell. Generallyspeaking, such a promoter might include either a human or viralpromoter.

In various instances, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used to obtain high-level expression of the geneof interest. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa gene of interest is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression of the gene product following transfection can beoptimized. Further, selection of a promoter that is regulated inresponse to specific physiologic signals can permit inducible expressionof the gene product. Several elements/promoters which may be employed,in the context of the present invention, to regulate the expression ofthe gene of interest are listed below. This list is not intended to beexhaustive of all the possible elements involved in the promotion ofgene expression but, merely, to be exemplary thereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Viral promoters, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid encoding a gene of interest in an expression construct. Someexamples of enhancers include Immunoglobulin Heavy Chain; ImmunoglobulinLight Chain; T-Cell Receptor; HLA DQ a and DQ b b-Interferon;Interleukin-2; Interleukin-2 Receptor; Gibbon Ape Leukemia Virus; MHCClass II 5 or HLA-DRa; b-Actin; Muscle Creatine Kinase; Prealbumin(Transthyretin); Elastase I; Metallothionein; Collagenase; Albumin Gene;a-Fetoprotein; a-Globin; b-Globin; c-fos; c-HA-ras; Insulin Neural CellAdhesion Molecule (NCAM); al-Antitrypsin; H2B (TH2B) Histone; Mouse orType I Collagen; Glucose-Regulated Proteins (GRP94 and GRP78); RatGrowth Hormone; Human Serum Amyloid A (SAA); Troponin I (TN I);Platelet-Derived Growth Factor; Duchenne Muscular Dystrophy; SV40 orCMV; Polyoma; Retroviruses; Papilloma Virus; Hepatitis B Virus; HumanImmunodeficiency Virus. Inducers such as phorbol ester (TFA) heavymetals; glucocorticoids; poly (rl)X; poly(rc); Ela; H₂ O₂ ; IL-1;Interferon, Newcastle Disease Virus; A23187; IL-6; Serum; SV40 Large TAntigen; FMA; thyroid Hormone; could be used. Additionally, anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of the gene. Eukaryoticcells can support cytoplasmic transcription from certain bacterialpromoters if the appropriate bacterial polymerase is provided, either aspart of the delivery complex or as an additional genetic expressionconstruct.

In certain instances, the expression construct will comprise a virus orengineered construct derived from a viral genome. The ability of certainviruses to enter cells via receptor-mediated endocytosis and tointegrate into the host cell genome and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (Ridgeway, 1988; Nicolas andRubenstein, 1988; Baichwal et al., 1986; Temin, 1986). The first virusesused as gene vectors were DNA viruses including the papoviruses (simianvirus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwalet al., 1986) and adenoviruses (Ridgeway, 1988; Baichwal et al., 1986).These have a relatively low capacity for foreign DNA sequences and havea restricted host spectrum. Furthermore, their oncogenic potential andcytopathic effects in permissive cells raise safety concerns. They canaccommodate only up to 8 kB of foreign genetic material but can bereadily introduced in a variety of cell lines and laboratory animals(Nicolas and Rubenstein, 1988; Temin, 1986).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Often, another element of the expressioncassette is a terminator. These elements can serve to enhance messagelevels and to minimize read through from the cassette into othersequences.

It is understood in the art that to bring a coding sequence under thecontrol of a promoter, or operatively linking a sequence to a promoter,one positions the 5' end of the transcription initiation site of thetranscriptional reading frame of the protein between about 1 and about50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter. Inaddition, where eukaryotic expression is contemplated, one will alsotypically desire to incorporate into the transcriptional unit (whichincludes the cotransporter protein) an appropriate polyadenylation site(e.g., 5'-AATAAA-3') if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides "downstream" of the termination site of the protein at aposition prior to transcription termination.

To date, there are no effective and specific ways of treating ordiminishing the growth of colorectal cancer. The background referencesdiscussed hereinabove are part of the present invention insofar as theyare applicable to the invention described herein.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide improvedmethods for the treatment of colorectal cancer. In fulfilling thisobject, there is provided a method for inhibiting the growth of gastrinexpressing colon cancer cells comprising reducing the gastrin expressionof the colon cancer cells by inducing the production of inhibitoryantisense polynucleotides in cancer cells.

In certain embodiments of the present invention there are providedmethods for treating colon cancer in a patient comprising the steps of(a) providing an expression construct comprising a promoter functionalin eukaryotic cells and a polynucleotide encoding a gastrin gene,wherein the polynucleotide is positioned antisense to and under thecontrol of the promoter; and (b) introducing the expression constructwithin the colon cancer in vivo to produce transformed colon cancercells deficient in gastrin production.

The colon cancer may be a human colon cancer. The expression constructpreferably is a viral vector, such as a retroviral vector, an adenoviralvector and an adeno-associated viral vector, with a retroviral vectorbeing most preferred. The promoter regions to be used in the presentinvention are known to those of skill in the art; for example, thepolynucleotide sequence may be under the control of CMV, LTR, SV40, orother strong tissue specific or bi-specific promoters currently beingdeveloped.

The method of introducing the expression construct within the coloncancer cells may comprise intratumoral injection, osmotic pump deliveryor targeted liposomal delivery. Of course these methods would have to beoptimized for the individual case using procedures known to those ofskill in the art. For optimizing the route of delivery, the efficiencyof infection/transfer will be determined by monitoring the expression ofa control vector that expresses a marker protein such as greenfluorescent protein (GFP) or β-Gal. Continuous perfusion of theexpression construct also is contemplated.

Also disclosed are compositions for treating colon cancer comprising apoly nucleotide sequence encoding a gastrin gene expression constructwherein the gastrin gene is positioned antisense to and under thecontrol of promoter functional in eukaryotic cells. The composition fortreating colon cancer may comprise a polynucleotide sequence of SEQ IDNO: 13 positioned antisense to and under the control of a promoter, or amodification thereof.

Another embodiment of the present invention includes treatment of humancolon cancer tumors, growing in vivo, by inoculating colon cancer cellsoverexpressing antisense gastrin RNA in the animals.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Retroviral vector (LNCX, a kind gift from Dr. Dusty Miller, FredHutchinson Cancer Research Center, Seattle) containing the gastrin cDNAin the anti-sense orientation (G-AS) and under the transcription controlof the cytomegalovirus (CMV) promoter is shown.

FIGS. 2A-2D. As shown in FIG. 2A, the human colon cancer cell linesColo-205A (sub-cloned from Colo-205 [ATCC]), Colo-320 (ATCC) and HCT-116were analyzed for relative concentrations of gastrin mRNA by RT-PCR(reverse transcriptase-polymerase chain reaction) using 2 μg of totalRNA in the reaction (Rx) mixture, as published previously (Xu et al.,1994; Singh et al., 1994a). Ethidium bromide staining of PCR products ofa representative Rx, using primers HG₄ (5'AGGCCCAGCCGTGGCACCACA3'; SEQID NO: 3) and HG₅ (5'TGGCTAGGCTCTGAAGCTTGGTT3'; SEQ ID NO: 4) is shown.The relative concentrations of gastrin mRNA in each cell line wereadditionally quantitated by competitive RT-PCR using gastrin gDNA as aninternal control as described previously (Xu et al., 1994; Singh et al.,1994a) and the data are presented in the text. Lanes 1-5=Mw markers,Colo-205A, HCT-116, Colo-320 and HCT-116 samples without RT,respectively. As shown in FIG. 2B, each cell line was transfected withC- or LNC-G-AS vector DNA and G418 resistant (250 ng/ml for HCT-116 and500 ng/ml for Colo-205A and Colo-320) colonies were selected (Singh etal., 1994b). As shown in FIGS. 2C and 2D, DNA was isolated from HCT-116and Colo-320 cells transfected with either the gastrin anti-sense vectorLNC-G-AS DNA (AS) or control (C) vector DNA.

FIGS. 3A-3B. The C and AS clones were plated at equal concentrations(6000/well) in 96-well plates. At the end of 7 days of growth in normalgrowth medium containing 0 to 10% FCS, the total number of viable cellswas determined by an MTT assay (Guo et al., 1990). The optical density(O.D.) of the Rx products was read at 540 nm by a microplate reader(Molecular Devices). Each bar represents mean±SEM of 18 observationsfrom 3 separate clones and is representative of 3-4 similar experiments.*=p<0.05 vs the respective control values.

FIG. 4. Expression of antisense (AS) gastrin RNA by AS retrovirus in thesupernatant of AS PA317 cells. PA317 clones transfected with eitherLNC-G-AS vector (AS-clones) or transfected with LNCX vector (controlclones) were drug selected and cultured for 2-3 days and supernatantcollected, as described in the text. The viral particles in thesupernatant were pelleted by centrifugation and processed for RNAextraction. The viral RNA thus extracted was analyzed for the presenceof either AS gastrin RNA transcripts (using the primers LNCX-1 and HG-4as described elsewhere herein) or neomycin transcripts by RT-PCR™. Theexpected sizes of the AS gastrin RNA fragment and neomycin fragmentswere obtained in the AS and C samples, respectively, as presented inLanes 1 and 3. Lane 5=molecular weight markers. Lanes 2 and 4 are blank.Arrow on the left indicates the presence of the AS gastrin transcript inAS samples. An arrow on the right shows the presence of neomycintranscript in the C samples. The C samples were negative for AS gastrinfragments as expected.

FIGS. 5A and 5B. Effect of Treating Colon Cancer Tumors Growing In Vivowith Retroviruses Expressing Antisense Gastrin RNA. HCT-116 tumorsgrowing in vivo in nude mice were intratumorally injected with eitherthe control (C) virus-32 or the antisense (AS) virus-32 as described inExample IV. At the end of the study, mice were decapitated and tumorsharvested from the two groups of mice treated with either the control orthe AS virus. Tumor weights were noted and presented as mean±SEM in theFIG. 5A. The same data is presented as a % change in tumor weights inFIG. 5B, wherein the average tumor weight in mice treated with the Cvirus is presented arbitrarily as 100%. On an average tumor weights inmice treated with the AS virus was reduced by ˜50% and the differencewas statistically significant. *=p<0.05 vs the control data.

FIGS. 6A and 6B. Effect of Treating Animals with Retroviruses ExpressingAS Gastrin RNA on Tumorigenesis of Colon Cancer Cells In Vivo. Athymicnude mice inoculated with HCT-116 cells were injected with either thecontrol(C)or antisense (AS)retrovirus in 3 separate groups as describedunder Example V. At the end of the study, mice were decapitated andnumber of tumors/animal group counted. The data is presented as numberof tumors measured/animal/group in FIG. 6A and is presented as % animalsthat develop the tumors in each group in FIG. 6B. The number oftumors/animal were significantly higher in the mice pre-treated with theC retrovirus compared to mice treated with either the AS-32 retrovirusor AS-42 retrovirus. *=p<0.05 vs the control data.

FIGS. 7A and 7B. Effect of Treating Colon Cancer Tumors Growing In Vivowith Retroviruses Expressing AS Gastrin RNA in the Presence or Absenceof Polybrene. Athymic nude mice bearing HCT- 116 tumors on either sideof the animal were injected (as described under Example VI) with eithermedium alone or medium containing polybrene or medium±polybrene in thepresence of either control virus (CV) or the antisense virus (AS-V) asshown in the figure. A group of animals received no injections. At theend of the study, mice were decapitated and tumor weights noted. Tumorweights (mean±SEM) in each group of animals is presented in FIG. 6A.Tumor weights measured in animals injected with the medium alone werearbitrarily assigned a 100% value, and tumor weights from all otheranimal groups are expressed as a % in FIG. 7A. Tumor weights weresignificantly reduced in mice injected with the AS virus in the presenceor absence of polybrene compared to that in C groups as shown.

FIGS. 8A-8F. Athymic nude mice were inoculated either bilaterally (FIGS.8A and 8D) or contralaterally (FIGS. 8B and 8E) with control (C) or wildtype (Wt) or antisense (AS) cells as shown. The cells were eitherpre-cultured in 1.0% FCS for 3 days (FIGS. 8A, 8B and 8C) or in 0.1% FCS(FIGS. 8D, 8E and 8F) before inoculation. After 21-30 days mice weredecapitated and tumor weights noted in the different groups of animals.The mean±SEM of tumor weights thus measured in the various groups ofanimals is presented in FIGS. 8A, 8B, 8D and 8E. The tumor weights thusmeasured in animals inoculated bilaterally with either the C clones ofHCT-116 cells or with the Wt HCT-116 cells were arbitrarily assigned a 0value and the % change in the tumor weights of the control and WtHCT-116 tumors in mice injected contralaterally with AS clones ispresented in FIGS. 8C and 8F. As can be seen from the data presented inFIGS. 8A and 8D, both the control (C-2) and wild type (Wt) HCT-116 cellsgrew rapidly and at the expected rate in mice that were inoculatedbilaterally with the C and Wt cells on both sides of the animals. The ASHCT-116 clones (AS-2 and AS-29) (that were expressing the AS gastrinmRNA) grew insignificantly in animals that were either bilaterallyinoculated with the AS cells (FIGS. 8A and 8D) or contralaterallyinoculated with the AS cells in the presence of the C or Wt cells (FIGS.8B and 8E). Importantly the tumorigenic growth of the C and Wt cells, invivo, was significantly suppressed in mice inoculated contralaterallywith the AS cells compared to that in mice inoculated bilaterally witheither C or Wt cells on both sides of the animals (compare the growth ofthe C-2 and Wt tumors in FIGS. 8B and 8E to that of C-2 and Wt tumors inFIGS. 8A and 8D, respectively). The latter fact can be furtherappreciated from data as presented in FIGS. 8C and 8F, wherein it can beseen that the growth of Wt and C-2 tumors was significantly reduced by40-60% in mice inoculated contralaterally with the AS clones (eitherAS-2 or AS-29), compared to that in mice inoculated bilaterally with Cor Wt cells. The growth of C-2 and Wt tumors was more severelysuppressed in mice inoculated with cells pre-cultured in 0.1% FCS (FIGS.8D-8F) compared to that in mice inoculated with cells pre-cultured in1.0% FCS (FIGS. 8A-8C).

FIGS. 9A and 9B. Mice were inoculated with either HCT-116 cells or withHT-29 cells (wild type cells), either bilaterally (FIG. 9A) orcontralaterally in the presence of HCT-116 AS-14 cells (FIG. 9B) asshown. Tumor weights of the HCT-116 Wt cells were significantly reducedwhen grown in mice inoculated contralaterally with the AS cells comparedto that in mice inoculated with Wt cells on both sides. The tumorigenicgrowth of a heterologous colon cancer cell line, HT-29, was even moresignificantly suppressed in the presence of the AS cells compared tothat in mice inoculated with HT-29 cells on both sides.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Colorectal cancers are a major cause of cancer related death in the U.S.There seems to be very little effective therapy for patients expressingsuch cancers other than dramatic surgery. The genetic events leading tothe development of colorectal cancer have been studied. However anunderstanding of molecular genetics of carcinogenesis, has not led topreventative or therapeutic measures. Advances in molecular geneticswill lead to better risk assessment and early diagnosis but colorectalcancers will remain a deadly disease for a majority of patients due tothe lack of an adjuvant therapy.

Ar least some colon cancers express gastrin and it is possible thatgastrin plays a role in the initiation of colon tumors. However, thefield is littered with conflicting reports as to the proliferativeeffects of gastrin and it processing intermediates in the development ofcolorectal cancers. There are, for example, reports suggesting thatgastrin gene products are not completely processed by colon cancers. Theprocessing intermediates of gastrin are thought to have a marked cellproliferating effect.

Other investigators have shown gastrin does not stimulate the growth inall colon cancer cells. Thus there appears to be conflicting data onwhether or not gastrin is involved in pathogenesis and the progressionof colon cancer. Despite the conflicting data in this area, the presentinventors contemplated that decreasing the gastrin expression may betherapeutically effective in diminishing the growth and/or inhibition ofcolon cancers.

Although there are antibodies available against gastrin, they arenon-specific and cross react with cholecystokinin. Furthermore,antibodies are not available against all precursor forms of gastrin.Even if an antibody that was specific for all forms of gastrin wasavailable, the large amounts of such an antibody that would be requiredto combat the effects of the gastrin gene products would make itstherapeutic use impractical. In addition, therapeutic antibodies areoften antigenic themselves.

The present inventors thus present an alternative that can be used forprophylaxis or treatment to inhibit growth of colon cancer cells. Theinventors have demonstrated, for the first time, a method forsignificantly inhibiting the growth of gastrin expressing colon cancercells, both in vivo and in vitro. There is provided herein a method forinhibiting the growth of gastrin expressing colon cancer cells, themethod comprising reducing the gastrin expression of the colon cancercells by inducing the production of inhibitory antisenseoligonucleotides in cancer cells. It will be understood with benefit ofthe present disclosure that one of skill in the art may, without undueexperimentation, devise protocols for routine screening ofoligonucleotide sequences for in vitro efficacy in the growth inhibitionof human colon cancer tumors. Further, with benefit of the presentdisclosure one of skill in the art may, having identifiedoligonucleotide sequences having in vitro efficacy in the growthinhibition human colon cancer tumors, fashion expression constructs fordelivery via any of several methods, including intratumoral injection,osmotic pump delivery, targeted liposomal delivery; alternatively, onemay inoculate patients having tumors with homologous tumor cells thatare transfected/infected ex vivo with the antisense gastrin RNAexpression vectors or viruses which may prove to be a highly specificand safe method of treating colon cancers growing at secondary sites inthe body. One of skill in the art may further develop human treatmentprotocols using the antisense gastrin identified as described above.Antisense gastrin treatment will be of use in the clinical treatment ofcolon cancers in which transformed or cancerous cells play a role. Suchtreatment will be particularly useful tools in anti-tumor therapy, forexample, in treating patients with colorectal cancers that are hormonedependent and mediated by gastrin or gastrin-like peptide expression.Other objects, features and advantages of the present invention willbecome apparent from the present disclosure, and it should be understoodthat various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

1. Gastrin

Gastrin is a peptide hormone produced by the G-cells of the gastricantrum. It controls acid secretion by the stomach and is believed toregulate growth of the normal intestinal mucosa. However, recentlyanother role for this peptide has emerged. It has been shown by thepresent inventors and others in the field that gastrin expression occursin cancer cell lines. A possible role of gastrin as an autocrine growthfactor for colon cancers is clearly different from its endocrine role asan acid secretion regulator and gastrointestinal muscosal growthregulator.

Many colon cancer cells express and secrete gastrin gene products (Daiet al., 1992; Kochman et al., 1992; Finley et al., 1993; Van Solinge etal., 1993; Xu et al., 1994; Singh et al., 1994a; Hoosein et al., 1988;Hoosein et al., 1990) and bind gastrin-like peptides (Singh et al.,1986; Singh et al., 1987; Weinstock and Baldwin, 1988; Watson andSteele, 1994; Upp et al., 1989; Singh et al., 1985) and it is possiblethat gastrin-like peptides serve as autocrine factors for colon cancers.In support of this, it is suggested that desensitization/intemalizationof gastrin receptors in response to endogenous gastrins may haverendered a majority of the colon cancer cells refractory(non-responsive) to exogenous gastrins in previous studies. Similarly inprevious reports gastrin antibodies were either reported to inhibit(Hoosein et al., 1988; Hoosein et al., 1990) or have no effect on thegrowth of colon cancer cell lines in vitro. However, we now know thatgastrin gene products are for the most part incompletely processed bycolon cancers, and processing intermediates (gly-gastrins andpro-gastrins) are the major forms expressed (Dai et al., 1992; Kochmanet al., 1992; Van Solinge et al., 1993; Singh et al., 1994a; Ciccotostoet al., 1995). While in the past C-terminal amidation of gastrin-likepeptides was considered a pre-requisite for measuring biologicaleffects, it has been reported that non-amidated gastrins (especiallygly-gastrins) exert proliferative effects on pancreatic cancer cells(Seva et al., 1994), fibroblasts, intestinal cells and colon cancercells (Dai et al., 1992; Singh et al., 1994a; Baldwin, 1995; Singh etal., 1995).

Throughout this specification the term "gastrin gene product" is used;as used herein it refers to the product of the full gastrin encodingpolynucleotide or any intermediates that may arise from the translationof the gastrin gene. "Processing intermediates" of gastrin are thosepeptides that may be derived through post translational modification ofthe gastrin gene, these include but are not limited to preprogastrin(SEQ ID NO: 14), progastrin (SEQ ID NO: 7), amidated gastrins (SEQ IDNO: 16 and SEQ ID:NO 17), glygastrin (SEQ ID NO: 15), and cryptogastrin(SEQ ID NO: 11).

In certain embodiments of the present invention it should be possible toinhibit the growth of colon cancer cells by presenting to the cellsantibodies derived against receptors of gastrin gene products. Anotheraspect of gastrin-stimulated colon cancer cell inhibition may involveantagonists of gastrin that would result in the inhibition of gastrinaction.

The nucleic acid (encompassed by SEQ ID NO: 3 and SEQ ID NO: 4) andcorresponding amino acid sequences of human gastrin have been elucidated(SEQ ID NO: 13 and SEQ ID NO: 14, respectively).

2. Antisense

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. The most effective antisense constructs include regionscomplementary to intron/exon splice junctions. One preferred embodimentincludes an antisense construct with complementarity to regions within50-200 bases of an intron-exon splice junction. It has been observedthat some exon sequences can be included in the construct withoutseriously affecting the target selectivity thereof. The amount of exonicmaterial included will vary, depending on the particular exon and intronsequences used. One can readily test whether too much exon DNA isincluded simply by testing the constructs in vitro to determine whethernormal cellular function is affected or whether the expression ofrelated genes having complementary sequences is affected.

As stated above, "complementary" or "antisense" means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme) could be designed. These molecules, though having lessthan 50% homology, would bind to target sequences under appropriateconditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

A preferred antisense sequence of the present invention is thecomplement to SEQ ID NO: 13, which is the antisense sequence of thegastrin cDNA. The experiments leading to this conclusion are outlinedbelow.

Phosphothiorated antisense oligonucleotides were synthesized. In initialexperiments both the sense and the antisense phosphothioratedoligonucleotides were observed to cause a significant decrease in theproliferation of colon cancer cells. It was suspected, however, that theoligonucleotides were possibly impure. Antisense phosphothioratedoligonucleotides were then purified. Only 20-30% growth inhibition ofcolon cancer cell lines was observed in the presence of the purifiedantisense phosphothiorated oligonucleotides. Since dramatic effects werenot observed with the antisense oligonucleotides, studies with theoriginal antisense oligonucleotides were discontinued. With the help ofthe GeneRunner computer program, new antisense oligonucleotides weredesigned new studies were conducted with them. Significant growthinhibitory effects were observed with the second batch ofoligonucleotides, although the inhibitory effects did notexceed >30-50%, suggesting that this route of administration may not beeffective for therapeutic purposes. Therefore studies with the antisenseoligonucleotides were abandoned and work began toward constructingantisense expression vectors as detailed herein.

Several sense and antisense primers were designed and prepared(designated HG1-HG6) to amplify cDNA fragments of human gastrin gene ofvarious sizes. By using various sets of sense and antisense primers, theinventors amplified cDNA fragments of various sizes and arbitrarilynamed them a-g. Fragments a and f were non-amplifiable, for reasons notcompletely understood. The inventors successfully amplified fragment gusing HG4 and HG3 primers (as the sense and antisense primers),respectively. But for reasons unknown this fragment was non-clonable.Fragments c, d and e were successfully amplified by RT-PCR™ and clonedinto expression vectors in the antisense orientation. Of these, thefragment d gave the best results. Several antisense oligonucleotidesthat overlapped the translation initiation site by 6 to 8 bases werealso synthesized. Unfortunately, none of these were very effective invitro, and these particular investigations with antisenseoligonucleotides were discontinued. Thus, after extensiveexperimentation with various fragments, the inventors finally usedfragment d for making the retroviral antisense constructs, as presentedelsewhere herein. The steps used to make the antisense construct in theretroviral vector (LNCX) are described below.

A retrovirus vector, LNCX, containing an internal human cytomegalovirus(CMB) promoter was used in construction of the gastrin antisense vector(LNC-g-AS) as diagrammatically presented in FIG. 1. Poly-A mRNA from ahuman colon cancer cell line, HCT-116, was reverse transcribed to cDNA.The full-length gastrin cDNA fragment that contained the entiregastrin-open reading frame and 44 bp of the non-translated 5' flanksequence was generated by PCR using the 5'primer HG₄ (the sequence forwhich was provided in the original application on page 13) and theantisense 3' primer HG₅ by published methods. PCR amplification of thecDNA with HG₄ (SEQ ID NO: 3) and HG₅ (SEQ ID NO: 4) resulted in theamplification of the human gastrin cDNA fragment d which includes partof SEQ ID NO: 13. The PCR product was subcloned into pSPF2 vector thatprovided unique restriction sites at the proximal ends of the cDNA (5',Clal; 3' Smal). The use of the restriction sites ensured the directionalcloning of the gastrin cDNA in the LNCX vector in an antisenseorientation and placed the cDNA under the transcription control of theCMV promoter (as diagrammatically represented in FIG. 1). The LNC-G-ASDNA was confirmed by restriction and DNA sequence analysis (as describedin FIG. 2).

3. In Vivo Delivery and Treatment Protocols

(a) Adenovirus

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. "Adenovirus expression vector" is meant toinclude those constructs containing adenovirus sequences sufficient tosupport packaging of the construct and to express an antisensepolynucleotide that has been cloned therein.

The inventors have generated gastrin antisense (AS) adenovirus that isexpressing AS gastrin RNA. A brief description of the method used tomake the AS adenovirus (designated AdAsGas) is provided in Example IXbelow. Testing the efficacy of infecting tumors with the AS gastrinadenovirus is currently contemplated.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization or adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP (located at 16.8 m.u.) is particularly efficient during the latephase of infection, and all the mRNA's issued from this promoter possessa 5'-tripartite leader (TL) sequence which makes them preferred mRNA'sfor translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure. Use of the YAC system in is an alternativeapproach for the production of recombinant adenovirus.

Generation and propagation of adenovirus vectors, which are replicationdeficient, depend on a unique helper cell line, designated 293, whichwas transformed from human embryonic kidney cells by AdS DNA fragmentsand constitutively expresses E1 proteins (Graham et al., 1977). Sincethe E3 region is dispensable from the adenovirus genome (Jones andShenk, 1978), the current adenovirus vectors, with the help of 293cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991).

In nature, adenovirus can package approximately 105% of the wild-typegenome (Ghosh-Choudhury et al., 1987), providing capacity for about 2extra kB of DNA. Combined with the approximately 5.5 kB of DNA that isreplaceable in the E1 and E3 regions, the maximum capacity of thecurrent adenovirus vector is under 7.5 kB, or about 15% of the totallength of the vector. More than 80% of the adenovirus viral genomeremains in the vector backbone and is the source of vector-bornecytotoxicity.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 Lsiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, it has historically been used for mostconstructions employing adenovirus as a vector and it is non-oncogenic.

As stated above, a typical vector is replication defective and will nothave an adenovirus E1 region. Thus, it will be most convenient tointroduce the polynucleotide encoding the gene of interest a theposition from which the E1-coding sequences have been removed. However,the position of insertion of the construct within the adenovirussequences is not critical to the invention. The polynucleotide encodingthe gene of interest may also be inserted in lieu of the deleted E3region in E3 replacement vectors as described by Karlsson et al. (1986)or in the E4 region where a helper cel line or helper virus complementsthe E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹ -10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus can be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

(b) Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5' and 3' endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a human cDNA, together with theretroviral LTR and packaging sequences is introduced into this cell line(by calcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

The inventors used the LNC-G-AS retroviral construct, describedelsewhere herein. The LNC-G-AS vector was packaged as a retrovirus inthe packaging cell line PA3 17, using the methods known to those ofskill in the art (see, for example, Ausubed et al. 1997). The controlLNCX vector (that does not contain the antisense gastrin fragment) wasalso packaged in PA31 7 cells to make the control retrovirus. Severalcolonies of LNC-G-AS virus and LNCX virus (control virus), were drugselected. The concentration of virus, secreted by several drug selectedcolonies, transfected with either LNCX vector or LNC-G-AS vector, wasdetermined by conducting colony forming assays (CFU/ML) using eitherNIH3T3 cells or HCT-116 cells, essentially as described in Ausubed etal. (1997). Of the various control (C) PA31 7 clones (transfected withLNCX vector), clone #32 was identified as the best producer clone with aCFU/mL of approximately 2.8×10⁶ by day three of cell plating. The viraltitre in the supernatants of AS-PA371 clones (that were transfected withthe LNC-G-AS vector) was similarly determined for many clones. The AS 32and AS42 clones were identified as the best producer clones with viraltitres of ˜1-6×10⁴ CFU/ML. The expression of antisense gastrin mRNA bythe AS32 and AS42 PA317 clones was confirmed by RT-PCR™ as shown in FIG.4. The viral supernatants from C32, AS32, and AS42 clones were used forinjecting into human colon cancer tumors growing in vivo, in nude micein three separate studies as described in Examples IV-VI.

Thus, even though the viral titer was not very high in the supernatantused for injections, and the mice were injected only 2-3 times over thecourse of tumorigenesis in vivo, the inventors obtained a significantsuppressive effect of the AS virus. Further, the inventors have recentlysubcloned the gastrin RNA fragment (flanked by SEQ ID NO: 3 at the 5'end and by SEQ ID NO: 4 at the 3' end, as described herein), in anadenoviral vector and confirmed its expression in an adenoviralpackaging cell line (see Example IX). The inventors contemplateexpressing the AS- and C- adenovirus at a high titer of 10×10¹⁰ PFU/ml,which may allow a much better response, since the adenovirus at the hightiter level can be expected to be at least 10-20 fold more efficient intransfecting the tumor cells, in vivo, which should result in inhibitingthe tumor growth even more significantly. An adenovirus expressing β-Galand GFP proteins is being used to confirm efficacy of infection in vivoby various routes.

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intact₋₋sequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

(c) Other viral vectors as expression constructs

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpes viruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

(d) Non-viral vectors

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. As described above, the preferred mechanism for deliveryis via viral infection where the expression construct is encapsidated inan infectious viral particle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal.,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or"episomes" encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In one embodiment, the expression construct may simply consist of nakedrecombinant DNA or plasmids. Transfer of the construct may be performedby any of the methods mentioned above which physically or chemicallypermeabilize the cell membrane. This is particularly applicable fortransfer in vitro but it may be applied to in vivo use as well. Dubenskyet al. (1984) successfully injected polyomavirus DNA in the form ofCaPO₄ precipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection. Benvenistyand Neshif (1986) also demonstrated that direct intraperitonealinjection of CaPO₄ precipitated plasmids results in expression of thetransfected genes. It is envisioned that DNA encoding a gene of interestmay also be transferred in a similar manner in vivo and express the geneproduct.

Another embodiment for transferring a naked DNA expression constructinto cells may involve particle bombardment. This method depends on theability to accelerate DNA coated microprojectiles to a high velocityallowing them to pierce cell membranes and enter cells without killingthem (Klein et al., 1987). Several devices for accelerating smallparticles have been developed. One such device relies on a high voltagedischarge to generate an electrical current, which in turn provides themotive force (Yang et al., 1990). The microprojectiles used haveconsisted of biologically inert substances such as tungsten or goldbeads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,ie., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990).

Recently, a synthetic neoglycoprotein, which recognizes the samereceptor as ASOR, has been used as a gene delivery vehicle (Ferkol etal., 1993; Perales et al., 1994) and epidermal growth factor (EGF) hasalso been used to deliver genes to squamous carcinoma cells (Myers, EPO0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type such as lung, epithelialor tumor cells, by any number of receptor-ligand systems with or withoutliposomes. For example, epidermal growth factor (EGF) receptor may beused as the receptor for mediated delivery of a nucleic acid encoding agene in many tumor cells that exhibit upregulation of EGF receptor.Mannose can be used to target the mannose receptor on liver cells. Also,antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA(melanoma) can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal., the delivery of a nucleic acid into the cells, invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues. Anderson et al., U.S. Pat. No.5,399,346, and incorporated herein in its entirety, disclose ex vivotherapeutic methods.

Presented in the examples which follow is evidence that homologous orheterologous colon cancer cells, thrasfected ex vivo with AS gastrinexpression vectors, suppress the growth of tumors in vivo, wheninoculated on the opposite side in mice. These novel studies aredescribed briefly below and in more detail in Example VII.

The inventors have demonstrated that growth of human colon cancertumors, in vivo, is significantly reduced by ˜40-60% in the presence ofantisense (AS) clones of colon cancer cells expressing the AS gastrinRNA. Mice were inoculated subdermally on the opposite sides with coloncancer cells in various combinations as follows: (i) HCT-116 control (C)clones (expressing the control vector) bilaterally (on both the left andright sides); (ii) HT-29 wild type (Wt) cells bilaterally; (iii) HCT-116Wt cells bilaterally; (iv) HCT-116 AS clones bilaterally; (v)contralaterally with HCT-116-C cells and HCT-116-AS cells on oppositesides; (vi) contralaterally with HCT-116-Wt cells and HCT-116-AS cellson opposite sides; and (vii) contralaterally with HT-29-Wt cells andHCT-116-AS cells on opposite sides. In mice inoculated with C or Wtcells bilaterally (groups i, ii, iii above), the tumors grew to theexpected size of 800-1000 mg each. In mice inoculated with the AS cellsbilaterally (group iv above), the tumors grew either insignificantly orto a maximum weight of 300 mg. In mice bearing the AS cells along withthe C or Wt cells contralaterally (groups v, vi, vii above), the weightof the C/Wt tumors was significantly reduced by ˜40-60% compared to thatin animals bearing the C or Wt tumors bilaterally (groups i, ii, iiiabove).

Importantly, the results of these studies for the first time indicatethat the presence of human colon cancer cells expressing the AS gastrinmRNA can result in negative bystander effects on the growth of the C/Wthuman colon cancer cells growing contralaterally on the opposite side ofthe animals. Investigations of the endocrine mechanisms that may bemediating these negative bystander effects suggest that the antisensecells secrete inhibitory factors (such as IGFBP-2), which may result ina significant reduction in the growth of colon cancer tumors on theopposite side of the animals.

(e) Pharmaceutical Compositions

Where clinical applications are contemplated, it will be necessary toprepare a pharmaceutical compositions--either gene delivery vectors orengineered cells--in a form appropriate for the intended application.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase"pharmaceutically or pharmacologically acceptable" refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, "pharmaceutically acceptable carrier" includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknow in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

Solutions of the active ingredients as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed withsurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent growth of microorganisms.

The expression vectors and delivery vehicles of the present inventionmay include classic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral., nasal., buccal., rectal., vaginal or topical.Alternatively, administration may be by orthotopic, intradermal.,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions, described supra.

The vectors, viruses, and cells of the present invention areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection also may beprepared. These preparations also may be emulsified. A typicalcomposition for such purposes comprises a 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters, suchas ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components in the pharmaceutical areadjusted according to well know parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical., the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on theintended goal. The term "unit dose" refers to a physically discrete unitsuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic composition calculated to produce thedesired response in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. Precise amounts of the therapeutic composition alsodepend on the judgment of the practitioner and are peculiar to eachindividual.

4. Kits

All the essential materials and reagents required for inhibiting tumorcell proliferation may be assembled together in a kit. This generallywill comprise selected expression vectors, viruses or cells. Alsoincluded may be various media for replication of the expression vectorsand host cells for such replication. Such kits will comprise distinctcontainers for each individual reagent.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being particularly preferred. For in vivo use,the expression vector, viruses, and cells may be formulated into apharmaceutically acceptable syringeable composition. In this case, thecontainer means may itself be an inhalant, syringe, pipette, eyedropper, or other such like apparatus, from which the formulation may beapplied to an infected area of the body, such as the colon, injectedinto an animal., or even applied to and mixed with the other componentsof the kit.

The components of the kit may also be provided in dried or lyophilizedforms. When reagents or components are provided as a dried form,reconstitution generally is by the addition of a suitable solvent. It isenvisioned that the solvent also may be provided in another containermeans.

The kits of the invention may also include an instruction sheet defining(i) administration of the antisense gastrin-expression vector construct;(ii) the antisense gastrin expressing viruses; and (iii) the antisensegastrin expressing cells.

A gastrin gene as used herein will be any contiguous segment of thepolynucleotide of SEQ ID NO: 13. This of course will include but is notlimited to the polynucleotide sequences for preprogastrin (SEQ ID NO:14), progastrin (SEQ ID NO: 7) amidated gastrins (SEQ ID NO: 16 and SEQID NO: 17), gly-gastrin (SEQ ID NO: 15) and cryptogastrin (SEQ ID NO:11).

The kits of the present invention also will typically include a meansfor containing the vials in close confinement for commercial sale suchas, e.g., injection or blow-molded plastic containers into which thedesired vials are retained. Irrespective of the number or type ofcontainers, the kits of the invention also may comprise, or be packagedwith, an instrument for assisting with the injection/administration orplacement of the ultimate complex composition within the body of ananimal. Such an instrument may be an inhalant, syringe, pipette,forceps, measured spoon, eye dropper or any such medically approveddelivery vehicle. Further, the temperatures at which the whole kits andparts thereof should be stored will be specified.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Expression of antisense gastrin cDNA into Colo-205A, Colo-320and HCT-116 colon cancer cell lines

A full length human gastrin cDNA was introduced in the antisensedirection (GAS) into a retroviral vector (LNCX) (Miller et al., 1993),and the recombinant vector (LNC-G-AS) (FIG. 1) was confirmed using DNAsequence analysis. The long terminal repeat (LTR) sequences, theneomycin phosphotransferase gene (neo) and restriction endonucleasesites utilized in the analysis in FIG. 2C are indicated. The retroviralvector is propagated in bacteria using a 2.3 kb vector DNA sequencederived from pBR322. B, BamHI; RV, EcoRV.

Three representative human colon cancer cell lines were selected basedon their relative levels of gastrin mRNA expression (FIG. 2A) andtransfected with either the control (LNCX vector) DNA (C) or theanti-sense (LNC-G-AS) vector DNA (AS). Total cellular RNA was analyzedby a competitive RT-PCR method for measuring relative concentrations ofgastrin mRNA/cell (Xu et al., 1994; Singh et al., 1994a). The Colo-205A, Colo-320 and HCT-116 cell lines expressed <0.5, -1-2 and 2-4copies/cell. Since Colo-205A cells expressed negligible amounts ofgastrin mRNA, this cell line served as a negative control.

The C and AS clones, numbered sequentially were expanded in vitro usingMccoy's SA (HCT-l 16) and RPMI-1640 (Colo-205A, Colo-320) growth medium(Gibco) in the presence of 10% fetal calf serum (FCS) (IrvineScientific). Total RNA was isolated (Narayan et al., 1992b) and analyzedfor expression of AS gastrin mRNA transcripts using a sense primerderived from the retroviral vector, LNCX (CCTGGAGACGCCATCCACGCT; SEQ IDNO: 5) (5' to the G-AS insert) and an antisense primer (HG₂) from theG-AS insert (GTGTATGTGCTGATCTTTGCACTG; SEQ ID NO: 6). A DNA fragment,consistent with the predicted size (482 bp), was present in all of theAS clones analyzed. None of the C clones were positive for the ASgastrin mRNA. Representative data from some of the C and AS clones fromthe 3 cell lines is presented. Lanes 1-8 for HCT-116=Mw markers, C₂,AS₁, AS₂, AS₃, AS₄, C₃ and Mw markers, respectively. Lanes 1-7 forColo-205A=Mw markers, C₁, C₃, C₄, AS₁, AS₂, and AS₃, respectively. Lanes1-5 for Colo-320=Mw markers, C₁, C₂, AS₂, and AS₃, respectively.

The cell lines were transfected with either the C or the AS vectors andG418 resistant colonies selected as described (Singh et al., 1994b). Theclones were expanded in growth medium containing 10% FCS under constantdrug selection. Of the five Colo-320-AS clones only one clone (AS₂)produced a sufficient number of cells to permit partial characterizationand analysis.

The Colo-320-AS₂ clone was analyzed using Southern hybridization andRT-PCR to ascertain the insertion and the expression of the LNC-G-ASvector (FIG. 2B and FIG. 2C). Southern analysis confirmed the presenceof one or more integrated copies of the LNC-G-AS proviral DNA in each ofthe LNC-G-AS transfectants (FIG. 2C). Genomic DNA (10μg) was digestedwith either BamHI (FIG. 2C) or EcoRV (FIG. 2D) using conditionsrecommended by the manufacturer (New England Biolabs) and analyzed byelectrophoresis on 0.75% agarose gels as described (Wood et al., 1994).

The results from EcoRV digests (FIG. 2D) suggest that all of theintegrated proviral DNAs in HCT-116-AS clones have not undergone arearrangement of the transfected retrovirus DNA. Hybridization analysiswas performed using a ³² P-dCTP labeled probe representing the entireopen reading frame of the gastrin cDNA. Lane 1, HCT-116-C₂ ; Lane 2,HCT-116-C₃ ; Lane 3, HCT-116; Lane 4, HCT-116-AS₂ ; Lane 5, HCT-116-AS₃; Lane 6, HCT-116-AS₆ ; Lane 7, Colo-320-C₃ ; Lane 8, Colo-320-AS₂.

The Colo-320-AS clone (FIG. 2D lane 8) contains at least two integratedcopies and one of these has undergone rearrangement. The endogenousgastrin gene is also detected in each of the DNA digests (4.9 kb, BamHI;8.8 kb, EcoRV).

Since the expression of gastrin AS RNA produced such a dramatic effectupon the proliferation of Colo-320 cells, it suggested for the firsttime that gastrin mRNA expression may indeed be critical to the growthof some colon cancer cell lines. Besides exhibiting an almost completegrowth arrest, the size of the Colo-320-AS cells was significantlyincreased (10-20 fold) compared to that of the control clones.

The Colo-320-AS clones demonstrated distinct morphological differencesunder the electron microscope (EM) compared to control clones. TheColo-320-AS cells were multi-nucleated with euchromatin and a highconcentration of mitochondria, while the Colo-320-C cells contained theexpected heterochromatin and few mitochondria. The Colo-320-AS cells,while clearly growth arrested, appeared to be metabolically active andexcluded Trypan blue dye. These distinct morphological differencesbetween the Colo-320-C and -AS clones may provide the first clues ofpossible intracellular mechanisms that may be mediating the mitogeniceffects of gastrin gene products in colon cancer cells.

EXAMPLE II Anti-proliferative effects of antisense gastrin RNA inHCT-116 cells

The anti-proliferative effects of expressing gastrin AS RNA in theHCT-116-AS cells were also analyzed. The in vitro proliferative rate ofthe HCT-116-C and AS clones was compared using an MTT assay (Guo et al.,1990). The proliferation of the HCT-116-AS clones in serum free medium(SFM) was 5% of that measured for HCT-116-C clones in SFM (FIG. 3). Theproliferation of the HCT-116-AS clones increased in response toincreasing concentrations of FCS, but remained lower than that ofHCT-116-C clones at equivalent concentrations of FCS (FIG. 3).

A soft agar clonogenic assay (Macpherson and Montagnier, 1964) was usedin order to determine the in vitro tumorigenic potential of the cells.The number of colonies formed by HCT-116-AS cells in increasingconcentrations of FCS, remained only 0-5% compared to that formed by theHCT-116-C clones. The in vitro tumorigenic potential for the C and ASclones of HCT-116 and Colo-205A cells was determined from a soft agarclonogenic assay (Macpherson and Montagnier, 1964). The cells wereseeded at equal concentrations (8000/well) in 6-well culture plates in0.3% agar in regular growth medium containing 0.1-10% FCS. The totalnumber of colonies/well were <1-5% in wells seeded with HCT-116-ASclones (at all concentrations of FCS) compared to wells seeded withHCT-116-C clones. Representative wells demonstrating total number ofcolonies formed by HCT-116-AS₂ and HCT- 116-C₂ clones after 14 days ofseeding in 1% FCS are shown.

The size of the colonies formed by HCT-116-AS vs HCT-116-C clones wasalso vastly different. The size and number of colonies formed by theColo-205A-AS and -C clones, was similar in all cases.. Thus while theproliferative potential of the HCT-116-AS clones (especially in 10% FCS)was not as drastically effected as that of the Colo-320-S clones, the invitro tumorigenic potential of the HCT-116-AS clones (even in 10% FCS)was significantly suppressed compared to that of the HCT-116-C andColo-320-C clones.

Morphologically, the HCT-116-AS clones also appeared to be significantlydifferent compared to the HCT-116-C clones. HCT-116-AS cells were -2-4fold larger in size, contained euchromatin, prominent nucleolus andsigns of microvilli formation; HCT-116-C cells appeared to be normalcolon cancer cells with typical heterochromatin.

EXAMPLE III In vivo anti-tumorigenic effects of expression of gastrinantisense RNA

To examine the in vivo anti-tumorigenic effects of expression of gastrinAS RNA nude mice tumor formation assays were conducted. Cells from theHCT-116-AS and -C clones were inoculated into nude mice (Narayan et al.,1992a; Singh et al., 1993). The mice were palpated for tumors from Day10.

The HCT-116-AS and C clones, enumerated in vitro in 1% FCS wereinoculated contralaterally at equal concentrations (0.1-0.5×10⁷cells/0.2 ml HBSS) in female nude (athymic) mice, age 2 months (LifeSciences, St. Petersburg, Fla.) following published procedures (Singh etal., 1986; Singh et al., 1987).

The mice were euthanized between Days 21-45 and tumors, free of hosttissues, removed (Singh et al., 1986; Singh et al., 1987) and tumorweights noted (Table 1). Each data point represents mean±SD of tumorweights from 2-3 mice inoculated with the indicated AS and controlclones growing contralaterally in mice; range of values measured isgiven in parenthesis. Either no or significantly smaller tumors wereobtained from the mice inoculated with HCT-116-AS clones (Table 1);tumors were palatable as early as Day 12 in the mice inoculated with theHCT-116-C clones. A well formed tumor was removed from every HCT-116-Cinoculation site at the time of euthanasia, and confirmed for vectorDNA.

The marked suppression of tumorigenesis of HCT-116-AS clones in vivoonce again suggests that gastrin gene products may play a critical rolein the growth and tumorigenesis of human colon cancer cells thatnormally are express significant concentrations of gastrin mRNA.

                  TABLE I                                                         ______________________________________                                        Tumorigenicity of HCT-116-AS and -C clones in vivo in nude mice                      Tumor Weights           Day of                                         Right Side       Left Side     Euthanasia                                            Clone   Weight    Clone Weight  (Post                                    Mice #  # (mgs) # (mgs) Inoculation)                                        ______________________________________                                        1,2,3  C.sub.2 639 ± 220                                                                            AS.sub.4                                                                            43.3 ± 75                                                                          37                                         (400-850)   (0.0-130)                                                       4,5,6 C.sub.3 534 ± 127 AS.sub.3 48.0 ± 59 37                             (397-650)   (0.0-114)                                                       7,8,9 C.sub.8 217 ± 89   AS.sub.10 1.5 ± 2 22                             (123-300)  (0.0-3.0)                                                        10,11 C.sub.7 417 ± 6  AS.sub.9   0.0 ± 0.0 22                            (413-422)  (0.0-0.0)                                                      ______________________________________                                    

EXAMPLE IV Inoculation of nude mice with wild type HCT-116 cells

Female or male nude mice (5 to 8 wk old) were subdermally inoculatedwith 5×10⁶ wild type HCT-116 cells on either side by the inventors'published methods (see Singh et al., 1996). Tumors were allowed to beestablished in the mice. By Day 10 post-inoculation of cells, ˜100 mgtumors were palpable in all the mice. The mice were divided into groupsof five, with each mice containing two tumors on either side growingsubdermally. Group 1 was injected with the control (C-32) viralsupernatant diluted to approximately contain 1×10⁵ CPU/ML. Group 2 wasinjected with the antisense virus (AS32). All the injections wereconducted intratumorally, wherein the animals were injected 0.1 ml ofthe viral supernatant, once a week (starting on Day 14 of tumor growth),for a total of two weeks. Tumors were harvested fthe inventors' daysafter the last injection, from either side of the animal., cleaned ofnecrotic and host tissues and weighed. Tumor weights thus measured inthe two groups of animals that were intratumorally injected either withthe control (C-32) virus or the antisense (AS-32) virus are presented inFIG. 5.

As can be seen from FIG. 5, intratumoral injection of the antisensevirus resulted in suppressing the growth of the tumors significantly(p<0.05) compared to the growth of the tumors that were injected withthe control virus. These results thus for the first time demonstratethat retroviruses, expressing antisense gastrin mRNA can significantlysuppress the growth of gastrin dependent human colon cancer tumors, invivo.

EXAMPLE V Rate of tumorigenesis was affected in the presence ofantisense virus

Athymic nude mice were inoculated with 5×10⁶ wild type HCT-116 cells,subdermally on either side of the animal as described in Example IV.Seven days post inoculation of the cells, at a time when the tumors wereyet not palpable in the mice, the mice were injected with either thecontrol (C-32) virus or the antisense (antisense-32) virus, at the siteof the cell injection, as described under Example IV. By injecting themice with the viral supernatants before the establishment of the tumors,the inventors wanted to examine if the rate of tumorigenesis wasaffected in the presence of antisense virus. The mice were injected 5×at intervals of 3-4 days and the number of palpable tumors/animal/groupdetermined. The results are presented in FIG. 6. As can be seen from thefigure, the group of mice that were injected with the antisense virus 32or 42, demonstrated a significant reduction in tumorigenesis; only 20%of the animals injected with the AS virus developed colon cancer tumorswhile 50% of the animals injected with the control virus developedtumors in the same time frame.

This example thus demonstrated that AS retrovirus not only suppressesthe growth of the tumors (as shown in FIG. 5), but also suppresses theinitiation/establishment of tumors (tumorigenesis).

EXAMPLE VI Improving the infection efficiency of retroviruses

Polybrene is used as an agent to improve the infection efficiency ofretroviruses. The inventors wanted to determine if the inclusion ofpolybrene in the inoculate would improve the efficacy of the AS virus.Animals in groups of 3-4 were inoculated with wild type HCT-116 cells oneither side of the animal as described under Example IV. Palpable tumors(approximately 100 mg each) were established in all animals by 10 dayspost inoculation of the colon cancer cells. Animals were injected fromDay 10 onwards with various combinations of either the antisense or thecontrol virus in the presence or absence of polybrene. A set of animalswere not injected at all. The animals received three injections, 3-4days apart, and tumors were harvested from the animals three days afterthe last injection. The results of the tumor weights measured in thevarious groups of animals are presented in FIG. 7. As can be seen fromthe figure, the co-injection of polybrene, itself resulted in slightsuppression of tumor growth (which was, however, not significant).Importantly the effectiveness of the antisense virus was similar in thepresence or absence of polybrene. These studies confirmed the fact thatinjection of well established human colon cancer tumors with virusesexpressing the antisense gastrin construct results in significantlysuppressing the growth of the human colon cancer tumors, in vivo, by˜30-50%.

EXAMPLE VII Gastrin-like Peptides in colon cancer cells

The inventors have demonstrated a significant retardation of in vitroand in vivo growth of antisense human colon cancer cells transfectedwith retroviral vectors expressing the antisense gastrin mRNA(LNC-G-AS), compared to the growth of control cells transfected with thecontrol [LNCX] vector (see Singh et al., 1996 and Examples I-IIIherein). In these examples, the inventors co-inoculated control andantisense clones on the opposite sides of test animals. It was noticedthat even though the growth of the control tumors was significantlyhigher than that of the antisense tumors, overall the growth of thecontrol tumors was less than the documented growth of thenon-transfected wild type cells. It was thius suspected that thepresence of the antisense cells in the same animals may have resulted inless than expected growth of the control clones. In order to test thispossibility several studies were conducted wherein the growth of eitherthe control HCT-116 cells or the wild type HCT-116 cells or aheterologous colon cancer cell line (HT-29) was recorded in mice thatwere either co-inoculated with the HCT-116 antisense clones or with thehomologous control cell line (either HCT-116-control clones or wild typeHCT116 cells or the HT-29 cells). The results of these studies arepresented in FIG. 8 and FIG. 9.

The results demonstrated for the first time that the co-presence ofantisense-human colon cancer cells expressing antisense gastrin mRNA,resulted in significantly suppressing the growth of homologous orheterologous human colon cancer cells, that were growing on the oppositeside of the mice. The presence of cells expressing the antisense gastrinmRNA within the same animal thus results in negative endocrine bystandereffects on the growth of the control tumors. The inventors are examiningthe mechanisms that may be mediating the negative bystander effects ofthe antisense cells, and the results so far indicate that the secretionof inhibitory factors, such as IGFBP-2, by the antisense cells may beresponsible for exerting the observed inhibitory effect on the growth ofhuman colon cancer cells growing elsewhere in the body of the mice.

It is therefore conceivable that colon cancers can be treated byinoculating the patients with homologous tumor cells that aretransfected/infected ex vivo with the antisense gastrin RNA expressionvectors or viruses which may prove to be a highly specific and safemethod of treating colon cancers growing at secondary sites in the body.

EXAMPLE VIII Gastrin-like Peptides in colon cancer cells

The concentrations of amidated gastrin and processing intermediates ofgastrin (pro-gastrin and gly-gastrin) (Varro et al., 1995; Nemeth etal., 1992a), in the cellular extracts (CE) and conditioned media (CM) ofthe HCT-116-AS and -C cells were also measured.

The concentration of gastrin-like peptides in the CM samples of ASclones was <1% compared to that in the CM of C clones (Table 2). Tworepresentative HCT-116-AS (AS₂, AS₃) and two representative HCT-116-C(C₂, C₃) clones were selected for analysis of gastrin-like peptides byRIA. The clones were expanded in vitro in 0.1 and 1% FCS. Cells werewashed in PBS, scraped with a rubber policeman, counted with a Coultercounter, and an equivalent number of cells (1×10⁸) suspended in 1 mldistilled water, boiled for 5 min, concentrated and de-salted using1000× cut-off amicon membranes. These samples were labeled CE. Anequivalent number of cells in duplicate 75 cm² flasks were processed forCM collection by our published procedures (Singh et al., 1994c) and theCM samples concentrated using the amicon concentrators. The CE and CMsamples were analyzed for pro-gastrin, gly-gastrin, and amidated gastrinusing specific antibodies L-289, L376 and L2, respectively, by RIA, aspublished previously (Varro et al., 1995; Nemeth et al., 1992a). Eachdata point represents fmoles/10⁷ cells and is the mean±SD of 4 separateobservations from 2 separate clones. The mean values for AS clones arealso presented as a percentage of the respective control values(arbitrarily assigned a 100% value).

The concentration of pro-gastrins and gly-gastrins was significantlyreduced in the CE samples of AS vs C clones. In contrast, a differencein the level of pp60 src-kinase protein (an unrelated protein expressedby colon cancer cells, Singh et al., 1994c and Singh et al., 1994d), wasnot observed in the CM samples of AS and C cells.

These results support the conclusion that the anti-proliferative effectsmeasured as a result of AS gastrin RNA expression were specific and dueto a significant reduction in the concentration of gastrin-likepeptides.

                  TABLE 2                                                         ______________________________________                                        Relative concentration of pro-gastrin and gly-gastrin in cellular             extracts                                                                        and conditioned media of HCT-116-C and -AS clones.                                   FCS Stimulation                                                                   0.1%            1.0%                                             Cell Line/clone                                                                        Pro-G     G-G       Pro-G   G-G                                      ______________________________________                                               Cellular Extract (CE) Samples                                          HCT-116-C                                                                              44.1 ± 2.0                                                                           22.6 ± 8.2                                                                           64.8 ± 9.3                                                                         23.6 ± 3.9                               (100.0) (100.0) (100.0) (100.0)                                              HCT-116-AS 15.5 ± 1.6  8.8 ± 0.3 25.0 ± 0   14.4 ± 0.7                                                 (35.1%) (38.9%) (38.5%) (61.0%)               Conditioned Media (CM) Samples                                         HCT-116-C                                                                              26.7 ± 3.0                                                                           22.3 ± 1.5                                                                           57.0    25.1 ± 3.6                              HCT-116-AS <1.0 <1.0 <1.0 <1.0                                              ______________________________________                                    

The proliferation and tumorigenic potential of the Colo-205A-AS and -Cclones was similar (FIG. 3), suggesting that the anti-proliferative andanti-tumorigenic effects of anti-sense expression of gastrin RNA werespecific to colon cancer cells expressing significant concentrations ofendogenous gastrin mRNA (Colo-320 and HCT-116 cells). No non-specificeffects were measured on either the morphology, tumorigenicity, orproliferation (FIG. 3) of Colo-205A cells expressing negligibleconcentrations of endogenous gastrin mRNA. The Colo-205A-AS clonesexpressed similar concentrations of AS gastrin RNA as the HCT-116-AS andthe Colo-320-AS clones (FIG. 2B), further confirming the specificity ofthe effects of anti-sense gastrin RNA expression on only the gastrinexpressing colon cancer cell lines.

Previous studies with antibodies suggested that gastrins may function asautocrine growth factors for colon cancers (Hoosein et al., 1988;Hoosein et al., 1990). The present inventors' studies confirm thatgastrin gene products may be novel autocrine growth factors for humancolon cancer cell lines that are expressing significant concentrationsof gastrin mRNA. The present inventors have demonstrated significantinhibition in the growth of Colo-320 cells with gastrin antisenseoligonucleotides (20-23 mer).

The present inventors results further demonstrate that expression of thegastrin AS RNA via either the retroviral construct (FIGS. 1-3), or viainfectious viral particles (FIGS. 5-7), will be effective in suppressingthe growth of human colon cancers expressing gastrin mRNA, when exposeddirectly to the vector/virus or when grown in the presence of cellsoverexpressing the vector/virus.

Based on current knowledge that perhaps >60-80% of human colon cancersexpress gastrin mRNA, delivery of gastrin AS RNA expression vectors to atumor site and transfection of the tumor cell will significantlysuppress the growth of gastrin expressing colon cancers. Tumor cells notexpressing gatsrin but stimulated by gastrin produced by other cellswould also be suppressed by antisense inhibition of gastrin productionby other cells, by negative bystander effects.

Colon cancers expressing a minimal concentration of gastrin mRNA are notas likely to respond to the anti-tumorigenic effects of gastrin AS RNAexpression and may perhaps represent a sub-set of tumors that havedeveloped autocrine mechanisms independent of gastrin gene products,unless they are dependent on gastrin produced by other cells that are,infact, transformed.

EXAMPLE IX Recombinant adenovirus AdAsGas

This example details the preparation of a recombinant adenovirusAdAsGas. The generation of recombinant adenovirus is based on homologousrecombination between the helper plasmid and the shuttle vector. Thesystems established by Bett et al. (1994) for the generation ofrecombinant adenovirus was used for making recombinant AdAsGas.

The DNA fragment, containing the antisense gastrin sequence, was clonedin front of CMV promoter in pCA4 shuttle vector. The presence of AsGassequences in this plasmid was confirmed by restriction mapping and DNAsequencing.

Both the helper plasmid pJM17, which has the complete Ad virus genomewith an insertion in the E1 region, and the pCA4-AsGas plasmid weregradient purified. Before transfection of low passage 293 cells(Microbix Biosystems, CA) the integrity of these two plasmids werechecked by restriction digest.

The virus generated using this system is replication competent on 293cells. Using a CellPhect kit from Pharmacia, the 293 cells weretransfected in triplicate with various ratios of helper to shuttleplasmid. The cells were overlayered with 1% soft agar containing media.After 8 days the plates were checked under a microscope for placqueformation and when positive they were overlayered with agarosecontaining neutral red. After overnight incubation at 37° C. and CO₂ theplaques were easy to observe without the microscope. Each plaque waspicked with a sterile pasture pipette and resuspended in 1 mL ofcomplete media in a sterile vial. Next 293 cells were infected with 100μL of the released virus from each vial. 48-96 h post infection,depending on when the cytopathic effects were observed, the cells wereharvested, virus released via freez-thaw, and viral DNA isolated bystandard methods. The presence of Wt Adenovirus as well as the presenceof AsGas were determined by PCR™.

All plaques which were collected showed absence of Wt Ad E1 region,indicating that the virus is indeed the recombinant AdAsGas virus.

EXAMPLE X Clinical trials of the use of antisense gastrin in treatingcolorectal cancer

This example is concerned with the development of human treatmentprotocols using the antisense gastrin. Antisense gastrin treatment willbe of use in the clinical treatment of colon cancers in whichtransformed or cancerous cells play a role. Such treatment will beparticularly useful tools in anti-tumor therapy, for example, intreating patients with colorectal cancers that are hormone dependent andmediated by gastrin or gastrin-like peptide expression.

The various elements of conducting a clinical trial., including patienttreatment and monitoring, are known to those of skill in the art inlight of the present disclosure. The following information is beingpresented as a general guideline for use in establishing antisensegastrin therapy in clinical trials.

Patients with advanced, colorectal cancers are appropriately selectedfor clinical study. Measurable disease is not required, however thepatient preferably has easily accessible pleural efflusion and/orascites. Further the patients most preferably carry tumors that expressgastrin or its processing intermediates. In an exemplary clinicalprotocol, patients may undergo placement of a Tenckhoff catheter, orother suitable device, into the site of the tumor. Typically, bonemarrow function, platelet count and renal function are measured todetermine the baseline cellularity.

The patient receives first a treatment of 10⁸ infectious particles ofadenovirus-antisense gastrin construct, diluted in sterile phosphatebuffered saline, via endoscopic intratumoral injections (total volume 1mL). Every three days the patient receives an identical treatment untila total of six treatments have been given. Other levels of constructdosages or administration protocols may be used to optimize desiredresults.

Three days after the sixth treatment, the tumor is examined to affirmthat it has decreased in size. Histological examinations should showconsiderable cell fragmentation at the tumor margin. Levels of gastrinproduction should have decreased markedly. A second course of sixtreatments is undertaken following which the tumor is further decreasedin size and is undergoing necrosis. The patient continues to receiveweekly treatments for three months or other lengths of time, after whichthe tumor should no longer be evident.

Alternatively, tumor cells are prepared from primary or secondary tumorsresected from the patients. The tumor cells from the patients aretransfected/infected with the AS gastrin RNA expression vectors/viruses,ex vivo, and confirmed to express the AS gastrin RNA. The AS gastrin RNAexpressing cells are inoculated back into the patients, subdermally, andthe patient monitored for metastatic disease. It is expected that thepresence of subdermally implanted/inoculated homologous AS cells willinhibit the tumorogenesis/growth of secondary tumors by negativebystander effects.

Of course, the above-described treatment regimes may be altered inaccordance with the knowledge gained from clinical trials that may beconducted as is routinely practiced by those of skill in the art. Thoseof skill in the art will be able to take the information disclosed inthis specification and optimize treatment regimes based on the clinicaltrials such as those described in this specification.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 17                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 613 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGCAGCGAC TATGTGTGTA TGTGCTGATC TTTGCACTGG CTCTGGCCGC CT -            #TCTCTGAA     60                                                                 - - GCTTCTTGGA AGCCCCGCTC CCAGCAGCCA GATGCACCCT TAGGTACAGG GG -            #CCAACAGG    120                                                                 - - GACCTGGAGC TACCCTGGCT GGAGCAGCAG GGCCCAGCCT CTCATCATCG AA -            #GGCAGCTG    180                                                                 - - GGACCCCAGG GTCCCCCACA CCTCGTGGCA GGTAGGAGCT GCTGACTGCC CT -            #GCTTGCCT    240                                                                 - - CACTTGGCCA GGTTTGGCCA AGGTCTCCCC AGACTGGCTC TGACTTCAGT TC -            #CTGGAAGG    300                                                                 - - TAGGCATCCT TCCCCCATTC TCGCCTCTCT CACCTCCTCA GACCCGTCCA AG -            #AAGCAGGG    360                                                                 - - ACCATGGCTG GAGGAAGAAG AAGAAGCCTA TGGATGGATG GACTTCGGCC GC -            #CGCAGTGC    420                                                                 - - TGAGGATGAG AACTAACAAT CCTAGAACCA AGCTTCAGAG CCTAGCCACC TC -            #CCACCCCA    480                                                                 - - CTCCAGCCCT GTCCCCTGAA AAACTGATCA AAAATAAACT AGTTTCCAGT GG -            #ATCAATGG    540                                                                 - - ACTGTGTCAG TGTTGTAGGG CAGAGGAGGG GGACTCATCT GGGGGTGAAG TT -            #GTGGCAGG    600                                                                 - - GAGAAGAGCT GAG              - #                  - #                      - #     613                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Gln Arg Leu Cys Val Tyr Val Leu Ile Ph - #e Ala Leu Ala Leu Ala      1               5   - #                10  - #                15               - - Ala Phe Ser Glu Ala Ser Trp Lys Pro Arg Se - #r Gln Gln Pro Asp Ala                  20      - #            25      - #            30                   - - Pro Leu Gly Thr Gly Ala Asn Arg Asp Leu Gl - #u Leu Pro Trp Leu Glu              35          - #        40          - #        45                       - - Gln Gln Gly Pro Ala Ser His His Arg Arg Gl - #n Leu Gly Pro Gln Gly          50              - #    55              - #    60                           - - Pro Pro His Leu Val Ala                                                  65                  - #70                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - AGGCCCAGCC GTGGCACCAC A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGGCTAGGCT CTGAAGCTTG GTT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CCTGGAGACG CCATCCACGC T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GTGTATGTGC TGATCTTTGC ACTG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ser Trp Lys Pro Arg Ser Gln Gln Pro Asp Al - #a Pro Leu Gly Thr Gly      1               5   - #                10  - #                15               - - Ala Asn Arg Asp Leu Glu Leu Pro Trp Leu Gl - #u Gln Gln Gly Pro Ala                  20      - #            25      - #            30                   - - Ser His His Arg Arg Gln Leu Gly Pro Gln Gl - #y Pro Pro His Leu Val              35          - #        40          - #        45                       - - Ala Asp Pro Ser Lys Lys Gln Gly Pro Trp Le - #u Glu Glu Glu Glu Glu          50              - #    55              - #    60                           - - Ala Tyr Gly Trp Met Asp Phe Gly Arg Arg                                  65                  - #70                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gln Leu Gly Pro Gln Gly Pro Pro His Leu Va - #l Ala Asp Pro Ser Lys      1               5   - #                10  - #                15               - - Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Gl - #u Ala Tyr Gly Trp Met                  20      - #            25      - #            30                   - - Asp Phe Gly                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Gln Leu Gly Pro Gln Gly Pro Pro His Leu Va - #l Ala Asp Pro Ser Lys      1               5   - #                10  - #                15               - - Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Gl - #u Ala Tyr Gly Trp Met                  20      - #            25      - #            30                   - - Asp Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Al - #a Tyr Gly Trp Met Asp      1               5   - #                10  - #                15               - - Phe Gly                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Ser Trp Lys Pro Arg Ser Gln Gln Pro Asp Al - #a Pro Leu Gly Thr Gly      1               5   - #                10  - #                15               - - Ala Asn Arg Asp Leu Glu Leu Pro Trp Leu Gl - #u Gln Gln Gly Pro Ala                  20      - #            25      - #            30                   - - Ser His His                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Pro Ser Lys Lys Gln Gly Pro Trp Leu Glu Gl - #u Glu Glu Glu Ala Tyr      1               5   - #                10  - #                15               - - Gly Trp Met Asp Phe                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 383 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - AGGCCCAGCC GTGGCACCAC ACACCTCCCA GCTCTGCAGG ACGAGATGCA GC -             #GACTATGT     60                                                                 - - GTGTATGTGC TGATCTTTGC ACTGGCTCTG GCCGCCTTCT CTGAAGCTTC TT -            #GGAAGCCC    120                                                                 - - CGCTCCCAGC AGCCAGATGC ACCCTTAGGT ACAGGGGCCA ACAGGGACCT GG -            #AGCTACCC    180                                                                 - - TGGCTGGAGC AGCAGGGCCC AGCCTCTCAT CATCGAAGGC AGCTGGGACC CC -            #AGGGTCCC    240                                                                 - - CCACACCTCG TGGCAGACCC GTCCAAGAAG CAGGGACCAT GGCTGGAGGA AG -            #AAGAAGAA    300                                                                 - - GCCTATGGAT GGATGGACTT CGGCCGCCGC AGTGCTGAGG ATGAGAACTA AC -            #AATCCTAG    360                                                                 - - AACCAAGCTT CAGAGCCTAG CCA           - #                  - #                   383                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Met Gln Arg Leu Cys Val Tyr Val Leu Ile Ph - #e Ala Leu Ala Leu Ala      1               5   - #                10  - #                15               - - Ala Phe Ser Glu Ala Ser Trp Lys Pro Arg Se - #r Gln Gln Pro Asp Ala                  20      - #            25      - #            30                   - - Pro Leu Gly Thr Gly Ala Asn Arg Asp Leu Gl - #u Leu Pro Trp Leu Glu              35          - #        40          - #        45                       - - Gln Gln Gly Pro Ala Ser His His Arg Arg Gl - #n Leu Gly Pro Gln Gly          50              - #    55              - #    60                           - - Pro Pro His Leu Val Ala Asp Pro Ser Lys Gl - #n Gly Pro Trp Leu Glu      65                  - #70                  - #75                  - #80        - - Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Ph - #e Gly Arg Arg Ser Ala                      85  - #                90  - #                95               - - Glu Asp Glu Asn                                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Al - #a Tyr Gly Trp Met Asp      1               5   - #                10  - #                15               - - Phe Gly                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Gln Leu Gly Pro Gln Gly Pro Pro His Leu Va - #l Ala Asp Pro Ser Lys      1               5   - #                10  - #                15               - - Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Gl - #u Ala Tyr Gly Trp Met                  20      - #            25      - #            30                   - - Asp Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Al - #a Tyr Gly Trp Met Asp      1               5   - #                10  - #                15               - - Phe                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated genetic construct comprising anantisense polynucleotide sequence of SEQ ID NO:13 flanked by SEQ ID NO:3 at the 5' end, and by SEQ ID NO:4 at the 3' end.
 2. The isolatedgenetic construct of claim 1 further comprising a promoter operativelylinked to the antisense polynucleotide.
 3. The isolated geneticconstruct of claim 1 which is a vector.
 4. The isolated geneticconstruct of claim 3 wherein the vector is a viral vector.
 5. Theisolated genetic construct of claim 3 wherein the vector is a retroviralvector.
 6. The isolated genetic construct of claim 3 wherein the vectoris an adenoviral vector or an adeno-associated viral vector.
 7. Theisolated genetic construct of claim 2 wherein the promoter is a CMV, LTRor SV40 promoter.
 8. A pharmaceutical composition comprising an isolatedgenetic construct comprising an antisense polynucleotide sequence of SEQID NO:13 flanked by SEQ ID NO: 3 at the 5' end, and by SEQ ID NO:4 atthe 3' end and a pharmaceutically acceptable carrier.